Amniotic Membrane Transplantation for Limbal Stem Cell Deficiency

Overview

The maintenance of a healthy functional corneal epithelium is provided by a unique subpopulation of stem cells located in the limbal region¹. When limbal epithelial stem cells are destroyed or become dysfunctional, a pathological state known as limbal stem cell deficiency (LSCD) manifests. The hallmark of LSCD is the conjunctivalization of the cornea², and is frequently associated with superficial vascularization and compromised corneal surface³.

LSCD can be found with a number of corneal diseases such as chemical burns, Stevens Johnson syndrome, aniridia, peripheral keratitis, severe limbitis, etc (for more details see Table 1). Patients with LSCD suffer from a severe loss of vision and annoying photophobia, and cannot be corrected by conventional PKP. Therefore, it is important to accurately diagnose LSCD because erroneous diagnosis may subject the patient to unnecessary surgeries. Correct diagnosis, which may require the use of impression cytology, will lead to reconstruction with limbal stem cell transplantation.

Corneal diseases with Limbal SC Deficiency

Destructive Loss of Stem Cells	Dysfunction of Limbal Niche
Chemical (mustard) or Thermal Burns Stevens Johnson Syndrome/TEN Multiple Surgeries or Cryotherapies to Limbus (latrogenic) Contact Lens-induced Keratopathy Severe Microbial Keratitis Anti-metabolite (5FU, MMC) Radiation	Aniridia / Iris Coloboma Multiple Endocrine Deficiency Chronic Limbitis or Peripheral Inflammatory / Ulcerative Disorders Neuronal or Ischemic Neurotropic Keratopathy Pterygium or Pseudopterygium Chronic Bullous Keratopathy Idiopathic
Opthalmology 102:1476, 1995; AJO 120:368, 1995 BJO 80:911, 1995; Cornea 7:31, 1998

Partial LSCD

Surgical Procedure for Treating Partial LSCD with Amniotic Membrane Transplantation (AMT)

For eyes with partial LSCD, where a part of the limbal circumference is damaged, the corneal surface can be reconstructed by debridement of conjunctivalized epithelium with4 or without5 transplantation of cryopreserved amniotic membrane as shown in Figure 1 and Video 1.

Partial LSCD presented as psuedopterygium and treated with excision and Amniotic Membrane Transplantation (AMT)

Surgial Steps:

1. Remove the corneal pannus via blunt superficial keratectomy
2. Polish the corneal surface with a dental burr (only if uneven)
3. Perform amniotic membrane transplantation using fibrin glue to cover the affected corneal surface and the bare sclera (as a Permanent graft)
4. Spare the visual axis if the remaining corneal stroma is smooth and clear centrally by opening a 6 mm diameter window on AM
5. If fibrin glue is not used, the cryopreserved amniotic membrane can be secured by several interrupted 10-0 nylon sutures on peri-limbal bulbar conjunctiva and by 8-0 Vicryl sutures in a mattress fashion, parallel to the fornix, with solid episcleral bites to seal the fornix border.
6. InsertPROKERA© to cover the cornea and the limbal area (as Temporary biologic graft). The benefits of cryopreserved amniotic membrane will be delivered through this sutureless conformer ring, which protects the cornea from external trauma while exerting biologic supportive functions to all covered parts of the ocular surface.

Total LSCD: Unilateral

Surgical Procedure for Treating Total Unilateral LSCD using a Conjunctival Limbal Autograft (CLAU) and Amniotic Membrane Transplantation (AMT)

For eyes inflected with total LSCD; where the entire limbal circumference is damaged, corneal surface reconstruction resorts to transplantation of limbal epithelial stem cells6. When total LSCD involves only one eye, successful reconstruction can be achieved by transplanting autologous limbal epithelial stem cells from the fellow eye in a procedure termed “conjunctival limbal autograft (CLAU)” as shown in Figure 2 and Video 2 below.7 Because the source of limbal epithelial stem cells is autologous, there is no risk of immune rejection and hence no need for systemic immunosuppression.

Unilateral total LSCD treated with superficial keratectomy AMT & CLAU

Surgical Procedure for CLAU and AMT

1. 1. Perform limbal peritomy
   2. Remove the corneal pannus via blunt superficial keratectomy
   3. Polish the corneal surface with a dental burr (only if uneven)
   4. Perform amniotic membrane transplantation using fibrin glue to cover the affected corneal surface and the bare sclera (as a Permanent graft – which allows the host cells to proliferate over the membrane). Spare the visual axis if the remaining corneal stroma is smooth and clear centrally by opening a 6 mm diameter window on AM
   5. Harvest a conjunctival limbal graft (CLAU) from the fellow eye consisting of a limbal arc length of 6 mm (about 60° of limbus), with 1 mm of peripheral cornea and 8 mm of the conjunctiva.11 [A recent report showed that one such strip is sufficient]
   6. Secure this free CLAU graft to the limbal area of the affected eye by interrupted 10-0 nylon sutures over the pre-placed amniotic membrane
   7. Cover the resulting bare area in the donor eye by cryopreserved amniotic membrane using fibrin glue
   8. Insert PROKERA® to cover the cornea and the limbal graft (as Temporary patch graft). The benefits of cryopreserved amniotic membrane will then be delivered through this sutureless conformer ring, which protects the cornea and the graft from external trauma while exerting biologic supportive functions to all covered parts of the ocular surface

Total LSCD: Bilateral

Surgical Treatment of Total Bilateral LSCD using a Keratolimbal Allograft (KLAL) and Amniotic Membrane Transplantation (AMT)

In cases of bilateral total LSCD, a keratolimbal allograft (KLAL) (Figure 2) is used and the success is dictated by effective immunosuppression8 as shown below in Figure 3 and Video 3.

Bilateral total LSCD treated with superficial keratectomy AMT & KLAL

Surgical Steps for Treating Bilateral LSCD using KLAL and AMT

1. Perform limbal peritomy
2. Remove the corneal pannus via blunt superficial keratectomy
3. Polish the corneal surface with a dental burr (only if uneven)
4. Perform amniotic membrane transplantation using fibrin glue to cover the affected corneal surface and the bare sclera (as a Permanent graft – which allows the host cells to proliferate over the membrane). Spare the visual axis if the remaining corneal stroma is smooth and clear centrally by opening a 6 mm diameter window on AM
5. Harvest a Keratolimbal allograft (KLAL) from a cadaveric donor and including 360° of the limbus, with 2-3 mm of peripheral cornea (obtained by a 7.5 to 8.0 mm trephine) and 0 to 5 mm of the sclera (depending the need for scleral repair or not)
6. Secure this free KLAL graft to the limbal area of the affected eye by interrupted 10-0 nylon sutures from the donor scleral edge to the recipient sclera (16 to 20 bites) through the underlying AM
7. Insert PROKERA® to cover the cornea and the limbal graft (as Temporary patch graft). The benefits of cryopreserved amniotic membrane will be delivered through this sutureless conformer ring, which protects the cornea and the graft from external trauma and while exerting biologic functions to all covered parts of the ocular surface

Other Considerations in Treating LSCD

For complex diseases associated with LSCD, it is important to restore the ocular surface’s defenses first so a stable tear film can be maintained before limbal stem cell transplantation (Figure 4). For eyes with conjunctival inflammation or cicatricial complications such as pathogenic symblepharon, it is important to correct these deficiencies before surgically transplanting limbal epithelial stem cells as described above.

Adjunctive use of amniotic membrane (AM) has beneficial effects which help preserve and expand the transplanted limbal stem cell population. These benefits include the restoration of an intact basement membrane which supports epithelial cell adhesion, differentiation, and migration⁹ while suppressing epithelial cell apoptosis.¹⁰

Because AM supports the growth of epithelial progenitor cells by prolonging their life span and maintaining their clonogenincity, 11 AM has also been used for ex vivo expansion of limbal stem cells,^{11, 12} as a sole treatment for partial LSCD,⁴ in acute stage of chemical burn¹³ and also as an adjunctive procedure in stem cell transplantation such as keratolimbal allograft (KLAL)⁸ and donor and/or recipient sites of conjunctival limbal autograft (CLAU)^{14, 15}. In these cases, AM not only promotes epithelialization, but also reduces inflammation, neovascularization and scar formation^{16, 17}.

 

 

Supplies for Treating LSCD with Amniotic Membrane Transplantation

Cryopreserved Amniotic Membrane

AMNIOGRAFT®, distributed by Bio-Tissue, Inc. (Miami, FL), is available in 4 sizes. The standard size to cover the cornea is 2.5 x 2.0 cm (Catalog # AG-2520). To cover only part of the cornea, the 2.0 x 1.5 cm (Catalog # AG-2015) can be used and if more than the cornea needs to be covered a 3.5 x 3.5 cm size (Catalog # AG-3535) can be used.

PROKERA®, distributed by Bio-Tissue, Inc., is a device that contains AMNIOGRAFT® clipped into a polycarbonate ring set conformer manufactured so the stromal side of the tissue is in contact with the corneal and limbal surface. This device can be inserted under the patient’s eye lids in the office or in the OR setting without the need for sutures or fibrin glue. There are two different diameters of PROKERA® available: 15 mm (Catalog # PK-15) and 16 mm (Catalog # PK-16). Most adult patients will tolerate a 16-mm device. After epithelial healing is completed, the membrane is dissolved and the PROKERA® ring set can be removed.

Fibrin Glue

Tisseel®, distributed by Baxter Biologics, Inc., is recommended over other fibrin glues because of the quick setting time. Tisseel® requires pre-warming in a thermal/stirrer provided without costs by the manufacturer. The recommended size is 2.0 mL (Catalog #: 1501236).

Sutures

Amniotic membrane transplantation is conventionally performed using sutures. If fibrin glue is not used, AMNIOGRAFT® can be secured using several interrupted 10-0 nylon sutures on peri-limbal bulbar conjunctiva and by 8-0 Vicryl sutures in a mattress fashion, parallel to the fornix, with solid episcleral bites to seal the fornix border.

Donor Limbal Tissue

Contact your local eye bank for more informaiton about receiving cadeveric tissue for LSCD surgery.

References

1. Cotsarelis G, Cheng SZ, Dong G, Sun T-T, Lavker RM. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201-209.
2. Puangsricharern V, Tseng SCG. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:1476-1485.
3. Dua HS, Jagjit SS, Azuara-Blanco A, Gupta P. Limbal stem cell deficiency: concept, aetiology, clinical presentation, diagnosis and management. Indian J Ophthalmol 2000;48:83-92.
4. Anderson DF, Ellies P, Pires RT, Tseng SC. Amniotic membrane transplantation for partial limbal stem cell deficiency. Br J Ophthalmol 2001;85:567-575.
5. Dua HS. The conjunctiva in corneal epithelial wound healing. Br J Ophthalmol 1998;82:1407-1411.
6. Tseng SCG, Prabhasawat P, Barton K, Gray T, Meller D. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch Ophthalmol 1998;116:431-441.
7. Kenyon KR, Tseng SC. Limbal autograft transplantation for ocular surface disorders. Ophthalmology 1989;96:709-722.
8. Espana EM, Di Pascuale M, Grueterich M, Solomon A, Tseng SC. Keratolimbal allograft in corneal reconstruction. Eye 2004;18:406-417.
9. Tseng SC, Tsubota K. Important concepts for treating ocular surface and tear disorders. Am J Ophthalmol 1997;124:825-835.
10. Boudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 1995;267:891-893.
11. Meller D, Pires RTF, Tseng SCG. Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures. Br J Ophthalmol 2002;86:463-471.
12. Nakamura T, Koizumi N, Tsuzuki M, Inoki K, Sano Y, Sotozono C, Kinoshita S. Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease. Cornea 2003;22:70-71.
13. Meller D, Pires RTF, Mack RJS, Figueiredo F, Heiligenhaus A, Park WC, Prabhasawat P, John T, McLeod SD, Steuhl KP, Tseng SCG. Amniotic membrane transplantation for acute chemical or thermal burns. Ophthalmology 2000;107:980-990.
14. Meallet MA, Espana EM, Grueterich M, Ti S-E, Goto E, Tseng SCG. Amniotic membrane transplantation for recipient and donor eyes undergoing conjunctival limbal autograft for total limbal stem cell deficiency. Ophthalmology 2003;110:1585-1592.
15. Kobayashi A, Shirao Y, Yoshita T, Yagami K, Segawa Y, Kawasaki K, Shozu M, Tseng SCG. Temporary amniotic membrane patching for acute chemical burns. Eye 2003;17:149-158.
16. Tseng SCG, Espana EM, Kawakita T, Di Pascuale MA, Wei Z-G, He H, Liu TS, Cho TH, Gao YY, Yeh LK, Liu C-Y. How does amniotic membrane work? The Ocular Surface 2004;2:177-187.
17. Dua HS, Gomes JA, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol 2004;49:51-77.

Videos

(none of the video links are working)